Compounds and methods of use for treatment of neurogenic inflammation

ABSTRACT

The subject invention pertains to compounds which are useful as anti-inflammatory agents and to compositions containing such compounds as active ingredients. The novel uses of the compounds relate to the anti-neurogenic inflammatory properties of the disclosed bis-heterocyclic compounds. Specifically exemplified herein are dragmacidin f, topsentin d, and topsentin e, and their salts, analogs, and derivatives.

CROSS-REFERENCE TO A RELATED APPLICATION

[0001] This application is a continuation of application Ser.No.09/253,118, filed Feb. 19, 1999; which claims priority to provisionalpatent application Ser. No. 60/091,990, filed Jul. 8, 1998; and Ser. No.60/075,476, filed Feb. 20, 1998.

DESCRIPTION

[0002] The subject invention was made with government support under aresearch project supported by NOAA Grant No. NA36RG0537. The governmenthas certain rights in this invention.

FIELD OF THE INVENTION

[0003] The subject invention pertains to compounds which are useful asanti-inflammatory agents and to compositions containing such compoundsas active ingredients. More particularly, the invention concerns novelbiologically active bis-heterocyclic compounds, e.g. bis-indoles, noveluses of the compounds, pharmaceutical compositions containing thesecompounds, and methods of producing the compounds. The novel uses of thecompounds relate to the anti-neurogenic inflammatory properties of thedisclosed bis-heterocyclic compounds. Specifically exemplified hereinare dragmacidin f, topsentin d and topsentin e, and their salts, analogsand derivatives.

BACKGROUND OF THE INVENTION

[0004] The prevention and control of inflammation is often of greatimportance for the treatment of humans and animals. Much research hasbeen devoted to development of compounds having anti-inflammatoryproperties. Certain methods and chemical compositions have beendeveloped which aid in inhibiting or controlling inflammation, butadditional anti-inflammatory methods and compositions are needed.

[0005] It has been found that some natural products and organisms arepotential sources for chemical molecules having useful biologicalactivity of great diversity. Marine sponges have proved to be such asource, and a number of publications have issued disclosing organiccompounds derived from marine sponges. Such publications includeScheuer, P. J. Ed. (1978-1983) Marine Natural Products, Chemical andBiological Perspectives, Academic Press, New York; Faulkner, D. (1995) JNat. Prod. Rep. 12:223-269; (1994) 11:355-394; (1993) 10:497-539; (1992)9:323-364; (1991) 8:97-147; (1990) 7:269-309; (1988) 5:613-663; (1987)4:539-576; (1986) 3:1-33; (1984) 1:551-598.

[0006] Indole compounds of marine origin have also been described inMoquin, C., M. Guyot (1984) Tetrahedron Letters 25 (44):5047-5048;Norton, R. S., R. J. Wells (1982) J Am. Chem. Soc. 104: 3628-3635; Roll,D. M., C. M. Iireland, H.S.M. Lu, J. Clardy (1988) J Org. Chem, 53:3276;Kobayashi, H., T. Ohta, S. Nozoe (1990) Tetrahedron 46:7699; Jiminez,C., E. Quinoa, P. Crews, (1991) Tetrahedron Lett. 32:1843; Jiminez, C.,E. QUinoa, M. Adamczeski, L. M. Hunter, P. Crews (1991) J Org. Chem.56:3403; Bifulco, G., I. Bruno, L. Minale, R. Riccio, A. Calignano, C.Debitus (1994) J Nat. Prod. 57:1294; and Bifulco, G., I. Bruno, R.Riccio, J. Lavayre, G. Bourdy (1995) J Nat. Prod. 58:1254.

[0007] Utilizing sponges as a source material and supplemented bysynthetic production methods, new classes of biologically activecompounds and new pharmaceutical compositions useful as antitumor andantiviral agents have been provided to the art. For example,bis-heterocyclic compounds such as bis-indoles have been previouslydescribed as having antimicrobial, antitumor or antiviral activity.Specifically, the bis-indole compounds known as topsentins are disclosedin U.S. Pat. No. 4,866,084. Dragmacidin and its related compoundsisolated from the marine sponge of the Dragmacidon sp. are disclosed inU.S. Pat. No.4,895,844. These patents are herein incorporated byreference. These compounds as well as the homocarbonyl topsentins andhamacanthins have also been described as having inhibitory activityagainst cellular inflammatory responses. See U.S. Pat. Nos. 5,290,777and 5,464,835, which are also hereby incorporated by reference. Thepresent invention provides additional related compounds which haveimproved water solubility characteristics and which have a novel utilityas anti-neurogenic inflammatory compositions.

[0008] Other advantages and further scope of applicability of thepresent invention will become apparent from the detailed descriptionsgiven herein; it should be understood, however, that the detaileddescriptions, while indicating preferred embodiments of the invention,are given by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent from such descriptions.

BRIEF SUMMARY OF THE INVENTION

[0009] The objects of the present invention are accomplished by theprovision of novel anti-inflammatory bis-heterocyclic compounds thathave a general structure according to the formula:

A₁—M—A₂

[0010] wherein each of A₁ and A₂is a heterocycle; and M is a core moietylinking the heterocycles, A₁ and A₂. In a specific embodiment, thecompound comprises an indole as the A₁ and A₂ moieties. Thus, thecompound can be a bis-indole. Other compounds of the subject inventioncan comprise heterocycles such as pyridyl or purine as the A₁ and A₂moieties. The core moiety M can be a linear or cyclic group comprisingat least three atoms.

[0011] Specifically exemplified herein are the novel compounds whichhave been designated as dragmacidin f, topsentin d, and topsentin e.Advantageously, these compounds have been found to possessanti-neurogenic inflammation activity.

[0012] As described herein, the invention also comprises pharmaceuticalcompositions, e.g. anti-neurogenic inflammatory compositions, containingas an active ingredient an effective amount, of one or more compoundsaccording to the formula expressed above and a non-toxic,pharmaceutically acceptable carrier or diluent. The pharmaceuticalcompositions of the subject invention can further comprise other activecompounds. Such other active compounds include, but are not limited to,anti-inflammatory compounds for example, steroidal compounds, includinghydrocortisone and the like; or non-steroidal anti-inflammatories,including acetylsalicylic acid (aspirin), ibuprofen, acetaminophen,indomethacin, and the like. The second active ingredient can includeantiviral, antibacterial, antifungal or other antimicrobial compounds orantitumor compounds as well.

[0013] As described herein, the invention further comprises processesfor the production of compounds and compositions of the invention andnovel methods of use thereof, e.g methods of inhibiting a neurogenicinflammatory response in a human or animal.

[0014] In accordance with the invention, methods for inhibitinginflammation comprise administering to a human or animal in need of suchtreatment an effective amount of the pharmaceutical composition

DETAILED DESCRIPTION OF THE INVENTION

[0015] The subject invention pertains to novel uses as anti-inflammatoryagents of bis-heterocyclic compounds and compositions comprising thebis-heterocyclic compounds. Surprisingly, the bis-heterocycle compoundsof the subject invention can be highly effective in inhibitingneurogenic inflammation.

[0016] A specific embodiment of the subject invention is a bis-indolecompound, wherein one of the indole rings has been reduced and aninternal cyclization has taken place between a2-aminoimidazolyl-ethylamine side chain and one of the indole rings asshown in structure (I) below:

[0017] R₁₋₄ are the same or different selected from —H, —OH, halogen,—R, —OR, —OCOR, —OA, or NZZ (wherein the Zs can be the same ordifferent); Y is the single group ═O, or the single group ═NZ, or twogroups, same or different, selected from —H, —OH, —OR, —OCOR, NZZ(wherein the Zs can be the same or different);

[0018] Z is independently selected from —H, —R, —OH, or —COR;

[0019] R is C1-C8 alkyl or C1-C8 alkoxyl, mesyl, or tosyl; and Ais-R-phenyl.

[0020] A particularly preferred embodiment is the compound calleddragmacidin f (structure (II), below), and salts thereof. In thiscompound, R₁═R₂═R₄═Z═H; R₃═Br; Y is the single group ═O.

[0021] A specific embodiment of the invention includes compounds inwhich alkylation of the imidazole ring of the topsentins has occured asshown in structure (III) below:

[0022] wherein

[0023] R₁-₈ are the same or different selected from —H, —OH, halogen,—R, —OR, —OCOR, —OA, or NZZ (wherein the Zs can be the same ordifferent);

[0024] R is C₁-₈ alkyl or aryl;

[0025] Z is independently selected from —H, —R, —OH, or —COR;

[0026] R is C₁-₈ alkyl or C₁-₈ alkoxyl, mesyl, or tosyl; and

[0027] A is —R—phenyl.

[0028] A particularly preferred embodiment is the compound in whichR₁═R₂═R₄═Z═H and R₃═Br and R₆═OH, and salts thereof, as shown below intopsentin d (IV):

[0029] A specific embodiment of the invention includes compounds inwhich alkylation of the imidazole ring of the topsentins has occurred asshown in structure (V) below:

[0030] wherein

[0031] R₁₋₈ are the same or different selected from —H, —OH, halogen,—R, —OR, —OCOR, —OA, or NZZ (wherein the Zs can be the same ordifferent);

[0032] R is C₁₋₈ alkyl or aryl;

[0033] Z is independently selected from —H, —R, —OH, or —COR;

[0034] R is C₁₋₈ alkyl or C₁₋₈ alkoxyl, mesyl, or tosyl; and

[0035] A is —R—phenyl.

[0036] A particularly preferred embodiment is the compound in whichR₁═R₂═R₄═Z═H and R₃═Br and R₆═OH, and salts thereof, as shown below intopsentin e (VI):

[0037] Skilled chemists having the benefit of the instant disclosure,can readily use procedures to prepare the subject compounds. In carryingout such operations, suitable filtration, chromatographic and otherpurification techniques can be used. These techniques could include, forexample, reversed phase (RPLC), column, vacuum flash, medium pressure(MPLC) and high performance liquid chromatography (HPLC) with a suitablecolumn such as silica gel, Sephadex LH-20, ammonia-treated silica gel,bonded phase RP-18, RP-8 and amino columns. Such columns are eluted withsuitable solvents such as heptane, ethyl acetate, methylene chloride,methanol, isopropanol, acetonitrile water, trifluoroacetic acid (TFA)and various combinations thereof. One method of preparation of thecompounds used according to the subject invention involves extractionfrom marine sponges of the genus Spongosorites (Phylum Porifera, ClassDemospongiae, Order Halichondrida, Family Halichondriidae). Certain ofthe samples used in connection with this invention have been assigned tothe species Spongosorites ruetzleri (HBOM Catalog Numbers 003:00112,003:00113, 003:00114, 003:00115, 003:00116, 003:00117, 003:00118,003:00119, 003:00120, 003:00927); other specimens represent new speciesof Spongosorites (HBOM Catalog Numbers 003:00549 and 003:00696). Fordescriptions of these samples and other Spongosorites species, refer toDiaz., M. C., Pomponi, S. A. and Van Soest, R. W. M. (1993) “Asystematic revision of the central West Atlantic Halichondrida(Demospongiae, Porifera), Part III: Description of valid species,”Scientia Marina, 57(4):283-306. All taxonomic voucher samples citedherein with HBOM Catalog Numbers are deposited in the Harbor BranchOceanographic Museum, Fort Pierce, Fla. All voucher specimens arepreserved in 70% ethanol with an expected shelf life of at least 30years and are accessible to those skilled in the art for taxonomicidentification purposes.

[0038] A novel use for the described compounds and compositions is theiradministration to an animal or human as an agent in the control of aneurogenic inflammatory response. Anti-inflammatory activity againstcellular activation of specific immune cells, e.g., phorbol myristateacetate (PMA)-induced inflammation, has been described for the subjectcompounds. See U.S. Pat. Nos. 5,290,777 and 5,464,835, which are herebyincorporated by reference. However, it is well recognized that activityof a compound in inhibiting cellular activated inflammation (e.g.,PMA-induced edema or inflammation) is not predictive or suggestive ofthat compound's activity in inhibiting neurogenic inflammation, e.g.,capsaicin-induced or resiniferatoxin (RTX)-induced edema orinflammation.

[0039] Therefore, the discovery that the subject compounds haveinhibitory activity against neurogenic inflammation is unexpected andadvantageous. Neurogenic inflammation is evoked by neuropeptidesreleased from primary afferent nerve terminals and by other secondarilyreleased inflammatory mediators. Specifically, neurogenic inflammationcan be evoked by neuropeptides, such as substance P (SP), calcitoningene-related peptide (CGRP), vasoactive intestinal peptide (VIP), andneurokinin A (NKA), released from primary afferent C-fiber nerveterminals and histamine, secondarily released from mast cells (Dray, A.,[1992] “Neuro pharmacological mechanisms of capsaicin and relatedsubstances” Biochem Pharm 44(4):611-15). For purposes of the subjectinvention, unless otherwise noted, the terms “inflammation” and“inflammatory response” refer to any and all such neurogenicinflammatory reactions including, but not limited to, immune-relatedresponses and/or allergic reactions to a physical, chemical, orbiological stimulus. “Anti-neurogenic inflammatory activity,” as usedherein, will be understood by those of ordinary skill in the art to meanbiological activity inhibiting or controlling a neurogenic inflammatoryresponse.

[0040] Anti-inflammatory activity can occur by modes of action which caninclude, but are not limited to, lipid-mediated inflammatory responses,e.g., (i) suppression of cellular activation of phospholipase A2, eitherdirectly (as is known for the anti-inflammatory compound, manoalide) orindirectly (as is known for the anti-inflammatory compound,hydrocortisone); (ii) by inhibiting, or controlling, cyclooxygenation ofarachidonic acid, similar to the action of non-steroidalanti-inflammatory drugs; or (iii) by affecting lipooxygenase products ofperoxidase reactions to arachidonic acid, or by non-lipid-mediatedinflammatory responses, e.g., protease-induced inflammatory responses,and the like. In addition, it is known that capsaicin (CAP), the activeconstituent found in cayenne pepper, induces an acute neurogenicinflammatory response when applied topically to skin. CAP is a highlyselective pain producing substance that selectively stimulatesnociceptive and thermal-sensitive nerve endings in tissues by acting ona specific membrane receptor. The mode of action of capsaicin thereforediffers significantly from phorbol myristate acetate (PMA)-inducedinflammation. By comparison, PMA elicits its pro-inflammatory effectsthrough cellular activation of specific immune cells, such asmacrophages and neutrophils. Consequently, the pain response to PMAdevelops more slowly than the immediate, but transient, pain response tocapsaicin.

[0041] The compounds and compositions of the subject inventionadvantageously can block the nociceptive (CAP-induced) inflammatorypathway, thereby providing a method for inhibiting neurogenicinflammation. Accordingly, the compounds can be used in the treatment ofinflammation at sites where the primary activating factor is ofneurogenic origin, e.g., inflammatory bowel disease or nepehritis. Thesubject compounds and compositions can be useful in the treatment ofchronic pain, migraines, thermal-induced pain, such as sunburn, or otherthermal and nociceptive pain, and chronic pain associated witharthritis. Uses can also include other inflammatory conditions thatinvolve a neurogenic pain-producing component, e.g., certain metasticcarcinomas or inflammation of the blood vessels.

[0042] The compounds of the subject invention can be used to treat avariety of skin conditions including, but not limited to, radiationirritation and burns (including UV and ionizing), chemical bums,rhinitis, thermal burns, reddening of the skin, and chemically induedlesions.

[0043] The compounds of the subject invention can also be used to treatallergic response and/or promote wound healing. This can include the useof the compounds in aerosol form for the treatment of acute allergicreactions such as acute asthmatic attack and in the treatment ofinflammation of the lung caused by chemical exposure.

[0044] The compounds of the subject invention can also be used to treatsystemic anaphylactic reactions in animals and man.

[0045] The compounds of the subject invention can also be used to treatconjunctivitis and inflammatory gum diseases.

[0046] Following are examples which illustrate procedures for practicingthe invention. A more complete understanding of the invention can beobtained by reference to the following specific examples of compounds,compositions, and methods of the invention. It will be apparent to thoseskilled in the art that the examples involve use of materials andreagents that are commercially available from known sources, e.g.,chemical supply houses, so no details are given respecting them. Theseexamples should not be construed as limiting. All percentages are byweight and all solvent mixture proportions are by volume unlessotherwise noted.

EXAMPLE 1

[0047] Preparation of Dragmacidin f (II)

[0048] Taxonomic and Collection Data.

[0049] A sample of Spongosorites sp. (Phylum Porifera, ClassDemospongiae, Order Halichondrida, Family Halichondriidae, HBOI no.22-VIII-90-3-004) was collected by dredge at a depth off 300 feet ofGran Canaria, Canary Islands (Latitude 27 45.75′ N, Longitude 15 47.90′W). This specimen of Spongosorites is spherical in shape andconsolidates sediment. A voucher specimen of the sponge has beendeposited at the Harbor Branch Oceanographic Museum, Catalog Number003:00156.

[0050] Isolation of Dragmacidin f (II).

[0051] The frozen sponge (35 g) was extracted exhaustively with ethanolby macerating in a Waring blender. The extract was filtered and thenconcentrated to a dark orange oil by distillation under reducedpressure. The residue was partitioned between ethyl acetate and water.The methanol soluble portion of the water partition was chromatographedunder vacuum column chromatographic conditions on an RP-18 stationaryphase. The column used had a volume of 360 ml and was 4 cm in height.The column was eluted with a step gradient ofacetonitrile-water-trifluoroacetic acid. The methanol soluble materials(797.8 mg) were applied to the column as a slurry adsorbed onto RP-18packing using water containing 0.1% trifluoroacetic acid (TFA). Thecolumn was eluted as follows: fraction 1, 100 mL of water containing0.1% TFA; fraction 2, 50 mL of water-ACN-TFA (40:10:0.05 v/v/v);fraction 3, 50 mL of water-ACN-TFA (30:20:0.05 v/v/v); fraction 4, 50 mLof water-ACN-TFA (20:30:0.05 v/v/v); fraction 5, 50 mL of water-ACN-TFA(10:40:0.05 v/v/v); fraction 6, 50 mL ACN. Fractions 2-4 were combined(179.6 mg) and chromatographed a second time under vcc conditions. Thecolumn (150 ml, 3 cm in height) was eluted as follows: fraction 1, 50 mLof water containing 0.1% TFA; fraction 2, 50 mL of water-ACN-TFA(40:10:0.05 v/v/v); fraction 3, 100 mL of water-ACN-TFA (30:20:0.05v/v/v); fraction 4, 50 mL of water-ACN-TFA (20:30:0.05 v/v/v); fraction5, 50 mL of water-ACN-TFA (10:40:0.05 v/v/v); fraction 6, 50 mL ACN.Fraction 3 was further separated by HPLC on a Vydac C-18 protein andpeptide column (4.6 mm×25 cm) using water-ACN-TFA (70:30:0.1) as eluent(flow rate=1 ml/min). Pure dragmacidin f, II, eluted after seven columnvolumes.

[0052] Spectral data:

[0053] HRFABMS: m/z 546.0821 C₂₅H₂₁BrN₇O₃ (calculated-observed=−0.2mmu).

[0054]¹³C NMR (125 MHZ, DMSO-d₆ trace of TFA); 14.89 q; 31.25 d; 34.69d; 43.52 t; 71.17 s; 111.79 s; 114.35 d; 114.87 s; 115.0 s; 121.24 s;123.09 d; 123.96 s; 124.29 d; 124.96 s; 126.94 s; 127.12 d; 127.13 d;130.69 s; 130.69 s; 131.15 d; 137.23 s; 147.78 s; 147.78 s;; 158.48 s;187.05 s.

[0055]¹H NMR (500 MHZ, DMSO-d₆ trace of TFA): 0.78 d (J═7.0 Hz); 2.29 dd(J═12.1, 2.4 Hz); 2.54 dd (J═12.1, 3.0 Hz); 3.29 m; 4.15 bs; 726 dd(J=8.6, 1.8 Hz); 7.54 bs; 7.54 d (J═2.9 Hz); 7.67 d (J═1.8 Hz); 8.55 d(J═8.6 Hz); 8.73 s; 11.67 s; 12.20 s; 12.39 s; 12.40 s.

EXAMPLE 2

[0056] Preparation of Topsentin D (III) and Topsentin E (IV)

[0057] Taxonomic and Collection data. A sample of Spongosorites sp.(Phylum Porifera, Class Demospongiae, Order Halichondrida, FamilyHalichondriidae, HBOI No. 21-V-93-3-001 was collected at a depth of 2067feet using the Johnson-Sea Link Submersible near Long Island, west ofCape Santa Maria, Bahamas (Latitude 23 41.117′ N Longitude 75 22.182′W). The sponge is thick encrusting with a yellow-white external colorand a yellow internal color. As voucher specimen has been deposited inthe Harbor Branch Oceanographic Museum (Catalog Number 003:00936).

[0058]

[0059] Isolation of topsentin d,(III).

[0060] The frozen sponge (250 g) was extracted exhaustively with ethanolby macerating in a Waring blender. The extract was filtered and thenconcentrated to a dark orange oil (15.9 g) by distillation under reducedpressure. The residue was partitioned between n-butanol and water. Thebutanol partition (4.1 g) was chromatographed under vacuum columnchromatographic conditions on an RP-18 stationary phase. The column usedhad a volume of 360 ml and was 4 cm in height. The column was elutedwith a step gradient of acetonitrile-water-trifluoracetic acid. Thematerials were applied to the column as a slurry adsorbed onto RP-18packing using water containing 0.1% trifluoroacetic acid (TFA). Thecolumn was eluted as follows; fraction 1,100 mLs of water containing 0.1% TFA; fraction 2,100 mLs of water-ACN-TFA (80: 20: 0.1 v/v/v); fraction3,100 mLs of water-ACN-TFA (60: 40: 0.1 v/v/v); Fraction 4,100 mLs ofwater-ACN-TFA (40: 60: 0.1 v/v/v); fraction 5,100 mLs of water-ACN-TFA(20:80: 0.1 v/v/v); fraction 6, 100 mLs ACN. Fraction 3 was furtherseparated by hplc on a Vydac C-18, protein and peptide column (1 cm x 25cm) using water-ACN-TFA (76: 24: 0.1) as eluent (flow rate=3 ml/min) toyield topsentin d (IV) 15.1 mg and topsentin e (VI) 10.4 mg.

[0061] Spectral data: Topsentin d (IV):

[0062] HRFABMS: 574.149 formula C₂₆H₂₁N₇O₄Br.

[0063]¹³C NMR (125 MHZ, d₆-DMSO+trace of TFA): 172.48 s, 172.25 s,155.52 s, 147.68 s, 142.68 s, 138.52 s, 137.77 d, 137.36 s, 131,55 s,128.1 d, 127.12 s, 126.71 s, 125.75 s, 123.13 d, 122.30 d, 121.37 d,119.08 s, 115.2 s, 115.05 d, 113.6 d, 110.77 d, 102.27 s, 98.29d,37.92t,29.60d.

[0064]¹H NMR (500 MHZ, d₆-DMSO+trace of TFA): 12.13 s, 12.01 s, 11.74 s(2 H), 8.43 d J═2 Hz, 8.02 d J═9 Hz, 7.68 s (2 H), 7.53 d J═9 Hz, 7.2 ddJ=9, 2 Hz, 6.92 d J=2 Hz, 6.79 dd J═9, 2 Hz, 4.55 m, 3.08 m (2 H).

[0065] Spectral data: Topsentin e (VI)

[0066] HRFABMS: 530.0942 Formula: C₂₅H2ON₇O₂Br.

[0067]¹³CNMR(125 MHZ, d₄-methanol): 172.8 s, 155.1 s, 147.9 s, 144.5 s,138.6 s, 137.6 s, 136.8 d, 136.8 s 129.9 s, 123.4 d,, 126.7 s, 126.6 d,122.5 d, 120.4 d, 120.0 s, 115.8 d, 114.7 d, 112.6 d, 126.0 s, 114.9 s,109.5 d, 103.9 s, 97.6 d, 28.7 d, 18.0 q.

[0068]¹H NMR (500 MHZ, d₄-methanol): 8.55 s, 8.13 d J═11.8 Hz, 7.65 s,7.51 s, 7.49 dd J═1 1.8, 2.5 Hz, 7.25 dd J═11.8, 2 Hz, 6.92 d J═2.5,6.87 bd J═1 1.8, 6.48 s, 4.30 q J═9.5 Hz, 1.69 d J═9.5 Hz.

EXAMPLE 3

[0069] Inhibition of Resiniferatoxin-Induced Inflammation (Edema) of theMouse Ear

[0070] Induction of mouse ear edema can be conducted by known methods(Inoue, 1-f., N. Nagata, Y. Koshffiara [1993]). Compounds to be testedfor anti-neurogenic inflammatory activity are topically applied inacetone to the ears of mice in a solution that includes theedema-causing irritant resiniferatoxin (RTX). RTX alone (0.1 μg/ear) orin combination with various dilutions of test compound are applied toboth sides of the left ears (5 mice per treatment group) and acetone isapplied to all right ears. After a 30-minute incubation, the mice aresacrificed, the ears removed, and bores taken and weighed. Edema ismeasured by subtracting the weight of the right ear (acetone control)from the weight of the left ear (treated). Results are recorded as %decrease (inhibition) or % increase (potentiation) in edema relative tothe control group edema.

[0071] Bis-heterocycle compounds of the subject invention, e.g., thebis-indole compounds, show significant anti-inflammatory properties.When screened for the ability to reduce edema in mouse ears caused byapplication of resiniferatoxin (RTX), a known inflammatory agent, a doseof 50 μg/ear of topsentin d, (IV), inhibited RTX-induced edema byapproximately 96.9%), and a dose of 50 μg/ear of topsentin e, (VI),inhibited RTX-induced edema by approximately 88.4%. In the same model, adose of 50 μg/ear of dragmacidin f, (II), inhibited RTX-induced edema byapproximately 69.9%.

EXAMPLE 4

[0072] Formulation and Administration

[0073] The compounds of the invention are useful for variousnon-therapeutic and therapeutic purposes. It is apparent from thetesting that the compounds of the invention are effective foranti-inflammatory uses.

[0074] Therapeutic application of the new compounds and compositionscontaining them can be contemplated to be accomplished by any suitabletherapeutic method and technique presently or prospectively known tothose skilled in the art. Further the compounds of the invention haveuse as starting material for intermediates for the preparation of otheruseful compounds and compositions.

[0075] In a preferred embodiment, the compounds or compositions ofthesubject invention are administered in a lotion or other cosmeticpreparation. This administration is done directly to the skin whereanti-inflammatory activity is desired.

[0076] The dosage administration to a host in the above indications willbe dependent upon the identity of the infection, the type of hostinvolved, its age, weight, health, kind of concurrent treatment, if any,frequency of treatment and therapeutic ration.

[0077] The compounds of the subject invention can be formulatedaccording to known methods for preparing pharmaceutically usefulcompositions. Formulations are described in detail in a number ofsources which are well known and readily available to those skilled inthe art. For example, Remington's Pharmaceutical Science by E. W. Martindescribes formulations which can be used in connection with the subjectinvention. In general, the compositions of the subject invention will beformulated such that an effective amount of the bioactive compound(s) iscombined with a suitable carrier in order to facilitate effectiveadministration of the composition.

[0078] In accordance with the invention, pharmaceutical compositionscomprising, as active ingredient, an effective amount of one or more ofthe subject compounds and one or more non-toxic, pharmaceuticallyacceptable carriers or diluents can be used by persons of ordinary skillin the art. In addition, the pharmaceutical composition can comprise oneor more of the bis-heterocycle compounds, e.g., a bis-indole, as a firstactive ingredient plus a second active ingredient comprising ananti-inflammatory compound known in the art. Such knownanti-inflammatory drugs include, but are not limited to, the steroidalanti-inflammatory drugs and the non-steroidal anti-inflammatory drugs(NSAIDs).

[0079] In accordance with this invention, pharmaceutically effectiveamounts of a known anti-inflammatory agent and the bis-heterocyclecompounds are administered sequentially or concurrently to the patient.The most effective mode of administration and dosage regimen ofbis-heterocycle compounds and anti-inflammatory agent will depend uponthe type of condition to be treated, the severity and course of thatcondition, previous therapy, the patient's health status, and responseto bis-indoles and the judgment of the treating physician.Bis-heterocycle compositions may be administered to the patient at onetime or over a series of treatments.

[0080] Preferably, the bis-heterocycle, e.g., a bis-indole composition,and any second anti-inflammatory agent are administered sequentially tothe patient, with the anti-inflammatory agent being administered before,after, or both before and after treatment with the bis-indole compound.Sequential administration involves treatment with the anti-inflammatoryagent at least on the same day (within 24 hours) of treatment withbis-indole and may involve continued treatment with theanti-inflammatory agent on days that the bis-indole is not administered.Conventional modes of administration and standard dosage regimens ofanti-inflammatory agents may be used (see Gilman, A. G. et. al. [eds]The Pharmacological Basis of Therapeutics, pp. 697-713, 1482, 1489-1491[1980]; Physicians Desk Reference, 1985 Edition). For example,indomethacin can be administered orally at a dosage of about 25-50 mg,three times a day. Higher doses can also be used. Alternatively, aspirin(about 1500-2000 mg/day), ibuprofen (about 1200-3200 mg/day), orconventional therapeutic doses of other anti-inflammatory agents can beused. Dosages of anti-inflammatory agents can be titrated to theindividual patient.

[0081] According to one embodiment of this invention, the patient mayreceive concurrent treatments with the anti-inflammatory agents andcompositions comprising bis-heterocycles, e.g. bis-indoles. For example,local intralesional, or intravenous injection of bis-indoles ispreferred (see Gilman et. al. supra at pp. 1290-91). Theanti-inflammatory agent should preferably be administered bysubcutaneous injection, subcutaneous slow release implant, or orally.

[0082] Alternatively, the patient can receive a composition comprising acombination of one or more bis-indole compounds and an anti-inflammatoryagent according to conventional modes of administration of agents whichexhibit antibacterial, anticancer, antitumor or anti-inflammatoryactivity. These include, for example, parenteral, subcutaneous,intravenous, or intralesional routes of administration.

[0083] The compounds used in these therapies can also be in a variety offorms. These include for example, solid, semi-solid and liquid dosageforms, such as tablets, pills, powders, liquid solutions or suspensions,suppositories, injectable and infusible solutions. The preferred formdepends on the intended mode of administration and therapeuticapplication. The compositions also preferably include conventionalpharmaceutically acceptable carriers and adjuvants which are known tothose of skill in the art. Preferably, the compositions of the inventionare in the form of a unit dose and will usually be administered to thepatient one or more times a day.

[0084] The compounds of the subject invention may also be administeredutilizing liposome technology, slow release capsules, implantable pumps,and biodegradable containers. These delivery methods can,advantageously, provide a uniform dosage over an extended period oftime.

[0085] Examples of such carriers or diluents include ethanol, dimethylsulfoxide, glycerol, silica, alumina, starch and equivalent carriers anddiluents. While effective amounts may vary, as conditions in whichcompositions are used vary, a minimal dosage required foranti-inflammatory activity is generally between 0.01 and 100 μg of thecompound. To provide for the administration of such dosages for thedesired therapeutic treatment, new pharmaceutical compositions of theinvention will advantageously comprise between about 0.1% and 45%, andespecially, 1 and 15% by weight of the total of one or more of the newcompounds based on the weight of the total composition including carrieror diluent.

[0086] Illustratively, dosage levels of the administered activeingredients can be: intravenous, 0.01 to about 20 mg/kg;intraperitoneal, 0.01 to about 100 mg/kg; subcutaneous, 0.01 to about100 mg/kg; intramuscular, 0.01 to about 100 mg/kg; orally 0.01 to about200 mg/kg and preferably about 1 to 100 mg/kg; intranasal instillation,0.01 to about 20 mg/kg; and aerosol, 0.01 to about 20 mg/kg of animal(body) weight.

[0087] Once improvement of the patient's condition has occurred, amaintenance dose is administered if necessary. Subsequently, the dosageor the frequency of administration, or both, may be reduced as afunction of the symptoms to a level at which the improved condition isretained. When the symptoms have been alleviated to the desired level,treatment should cease. Patients may however require intermittenttreatment on a long-term basis upon any recurrence of disease symptoms.

[0088] It should be understood that the examples and embodimentsdescribed herein are for illustrative purposes only and that variousmodifications or changes in light thereof will be suggested to personsskilled in the art and are to be included within the spirit and purviewof this application and the scope of the appended claims.

1. A method of treating inflammation in a human or animal, wherein saidmethod comprises administering to said human or animal an effectiveamount of a compound having the following formula: A₁—M—A₂ wherein eachof A₁ and A₂ is a heterocycle; and M is a core moiety linking A₁ and A₂.2. The method, according to claim 1, wherein said compound has thefollowing structure:

R₁₋₄ are the same or different and are selected from the groupconsisting of —H, —OH, halogen, —R, —OR, —OCOR, —OA, and NZZ (whereinthe Zs can be the same or different); Y is the single group ═O, or thesingle group ═NZ, or two groups, same or different, selected from thegroup consisting of —H, —OH, —OR, —OCOR, and NZZ (wherein the Zs can bethe same or different); Z is independently selected from the groupconsisting of —H, —R, —OH, and —COR; and R is C1-C8 alkyl or C1-C8alkoxyl, mesyl, or tosyl; and A is —R—phenyl.
 3. The method, accordingto claim 2, wherein said compound has the following structure:


4. The method, according to claim 1, wherein said method is used totreat inflammation in which the primary activating inflammation is ofneurogenic origin.
 5. The method, according to claim 4, wherein saidmethod is used to block a nociceptive inflammatory pathway.
 6. Themethod, according to claim 1, wherein said compound is administered as apharmaceutical composition, said pharmaceutical composition comprisingone or more compounds of claim 1 and an acceptable pharmaceuticalcarrier.
 7. The method, according to claim 1, wherein said compound isadministered as a cosmetic composition.
 8. An anti-inflamatorycomposition comprising a compound having the formula: A₁—M—A₂ whereineach of A₁ and A₂ is a heterocyclic; and M is a core moiety linking A₁and A₂; wherein said composition further comprises a pharmaceuticallyacceptable carrier.
 9. The composition, according to claim 8, whereinsaid compound has the following structure:

R₁₋₄ are the same or different and are selected from the groupconsisting of —H, —OH, halogen, —R, —OR, —OCOR, —OA, and NZZ (whereinthe Zs can be the same or different); Y is the single group ═O, or thesingle group ═NZ, or two groups, same or different, selected from thegroup consisting of —H, —OH, —OR, —OCOR, and NZZ (wherein the Zs can bethe same or different); Z is independently selected from the groupconsisting of —H, —R, —OH, and —COR; and R is C1-C8 alkyl or C1-C8alkoxyl, mesyl, or tosyl; and A is —R—phenyl.
 10. The composition,according to claim 9, wherein said compound has the following structure:


11. The composition, according to claim 8, wherein said compositionfurther comprises a second active ingredient.
 12. The composition,according to claim 11, wherein said second active agent is selected fromthe group consisting of a steroidal anti-inflammatory compound, anon-steroidal anti-inflammatory compound, an antiviral compound, anantibacterial compound, an antifungal compound, and an anti-tumorcompound.
 13. The composition, according to claim 8, wherein saidcompound is about 0.1% to about 45% weight percent of said composition.14. The composition, according to claim 13, wherein said compound isabout 1% to about 25%, weight percent, of said composition.
 15. Acompound having the following structure:

wherein R₁₋₄ are the same or different and selected from the groupconsisting of —H, —OH, halogen, —R, —OR, —OCOR, —OA, and NZZ (whereinthe Zs can be the same or different); Y is the single group ═O, or thesingle group ═NZ, or two groups, same or different, selected from thegroup consisting of —H, —OH, —OR, —OCOR, and NZZ (wherein the Zs can bethe same or different); Z is independently selected from the groupconsisting of —H, —R, —OH, and —COR; R is C1-C8 alkyl or C1-C8 alkoxyl,mesyl, or tosyl; and A is —R—phenyl.
 16. The compound, according toclaim 15, wherein said compound has the following structure:


17. A method for treating a condition, in a human or animal, whereinsaid condition is selected from the group consisting of pain, burns,allergic responses, wound healing, anaphylactic reactions, inflammatorybowel disease, nephritis, conjunctivitis, inflammatory gum disease, andacute asthmatic attack and inflammation of the lung due to chemicalexposure, said method comprising administering an effective amount of abis-heterocyclic compound or a salt, analog, or derivative thereof,wherein said compound is selected from the group consisting of

R₁₋₄ are the same or different and are selected from the groupconsisting of —H, —OH, halogen, —R, —OR, —OCOR, —OA, and NZZ (whereinthe Zs can be the same or different); Y is the single group ═O, or thesingle group ═NZ, or two groups, same or different, selected from thegroup consisting of —H, —OH, —OR, —OCOR, and NZZ (wherein the Zs can bethe same or different); Z is independently selected from the groupconsisting of —H, —R, —OH, and —COR; and R is C1-C8 alkyl or C1-C8alkoxyl, mesyl, or tosyl; and A is —R—phenyl;

wherein R₁₋₈ are the same or different and are selected from the groupconsisting of —H, —OH, halogen, —R, —OR, —OCOR, —OA, and NZZ (whereinthe Zs can be the same or different); R₉ is selected from the groupconsisting of C1-8 alkyl and aryl; Z is independently selected from thegroup consisting of —H, —R, —OH, or —COR; R is selected from the groupconsisting of C₁₋₈ alkyl or C₁₋₈ alkoxyl, mesyl, or tosyl; and A is—R—phenyl; and

wherein R₁₋₈ are the same or different and are selected from the groupconsisting of —H, —OH, halogen, —R, —OR, —OCOR, —OA, and NZZ (whereinthe Zs can be the same or different); R₉ is selected from the groupconsisting of C₁₋₈ alkyl and aryl; Z is independently selected from thegroup consisting of —H, —R, —OH, or —COR; R is selected from the groupconsisting of C₁₋₈ alkyl or C₁₋₈ alkoxyl, mesyl, and tosyl; and A is—R—phenyl.
 18. The method, according to claim 17, wherein said pain iscaused by a condition selected from the group consisting of migraine,rhinitis, thermal induced pain, radiation induced pain, and chemicalinduced pain.
 19. The method, according to claim 17, wherein said burnis selected from the group consisting of chemical burns, chemicalinduced lesions, radiation bums, and thermal bums.
 20. The method,according to claim 17, wherein said condition is allergic response. 21.The method, according to claim 17, wherein said treatment facilitatesthe promotion of wound healing.
 22. The method, according to claim 17,wherein said condition is a systemic anaphylactic reaction in an animalor a human.
 23. The method, according to claim 17, wherein saidcondition is inflammatory bowel disease.
 24. The method, according toclaim 17, wherein said condition is nephritis.
 25. The method, accordingto claim 17, wherein said condition is conjunctivitis.
 26. The method,according to claim 17, wherein said condition is inflammatory gumdisease.
 27. The method, according to claim 17, wherein said conditionis selected from the group consisting of acute asthmatic attack andinflammation of the lung from chemical exposure.